Patricia L. Foley

IL-17A Inhibits the Expansion of IL-17A-Producing T Cells in Mice through “Short-Loop” Inhibition via IL-17 Receptor

IL-23 and IL-17A regulate granulopoiesis through G-CSF, the main granulopoietic cytokine. IL-23 is secreted by activated macrophages and dendritic cells and promotes the expansion of three subsets of IL-17A-expressing neutrophil-regulatory T (Tn) cells; CD4−CD8−αβlow, CD4+CD8−αβ+ (Th17), and γδ+ T cells. In this study, we investigate the effects of IL-17A on circulating neutrophil levels using IL-17R-deficient (Il17ra−/−) mice and Il17ra−/−Itgb2−/− mice that lack both IL-17R and all four β2 integrins. IL-17R deficiency conferred a reduction in neutrophil numbers and G-CSF levels, as did Ab blockade against IL-17A in wild-type mice. Bone marrow transplantation revealed that IL-17R expression on nonhemopoietic cells had the greatest effects on regulating blood neutrophil counts. Although circulating neutrophil numbers were reduced, IL-17A expression, secretion, and the number of IL-17A-producing Tn cells were elevated in Il17ra−/− and Il17ra−/−Itgb2−/− mice, suggesting a negative feedback effect through IL-17R. The negative regulation of IL-17A-producing T cells and IL-17A and IL-17F gene expression through the interactions of IL-17A or IL-17F with IL-17R was confirmed in splenocyte cultures in vitro. We conclude that IL-17A regulates blood neutrophil counts by inducing G-CSF production mainly in nonhemopoietic cells. IL-17A controls the expansion of IL-17A-producing Tn cell populations through IL-17R.

Reversal of subarachnoid hemorrhage-induced vasoconstriction with an endothelin receptor antagonist.

Increased concentrations of the vasoconstrictor endothelin have recently been demonstrated in the cerebrospinal fluid after subarachnoid hemorrhage (SAH). This observation is consistent with the hypothesis that SAH-induced vasopasm is mediated in part by enhanced constriction due to endothelin. To investigate the issue, an endothelin receptor antagonist (ETant), cyclo(D-Asp-L-Pro-D-Val-L-Leu-D-Trp), was tested for its ability to reverse vasoconstriction after SAH. A transclival surgical approach to the basilar artery in rabbits was used, and the arterial diameter was measured continuously by videomicroscopy. Rabbits were divided randomly into six groups: 1) normal rabbits treated with 40 nmol/L ETant only; 2) normal rabbits treated with 50 mmol/L KCl, then 50 mmol/L KCl + 40 nmol/L ETant; 3) normal rabbits treated with 20 nmol/L endothelin-1 (ET-1), then 20 nmol/L ET-1 + 40 nmol/L ETant; 4) rabbits treated with 20 nmol/L ET-1 only; 5) rabbits subjected to SAH and treated with 40 nmol/L ETant; and 6) rabbits subjected to SAH and treated with artificial cerebrospinal fluid only. In normal (non-SAH) rabbits, ETant: 1) had little or no effect on resting tone; 2) did not reverse potassium-induced constrictions; and 3) substantially reversed endothelin-induced constrictions. The diameter of normal rabbit basilar arteries was 832.1 +/- 20.0 microns (mean +/- standard error). After SAH (double hemorrhage model), the mean diameter was 517.4 +/- 18.3 microns. The addition of ETant reversed this SAH-induced constriction by 70.7%.(ABSTRACT TRUNCATED AT 250 WORDS)

Alignment and composition of laminin–polycaprolactone nanofiber blends enhance peripheral nerve regeneration

Peripheral nerve transection occurs commonly in traumatic injury, causing deficits distal to the injury site. Conduits for repair currently on the market are hollow tubes; however, they often fail due to slow regeneration over long gaps. To facilitate increased regeneration speed and functional recovery, the ideal conduit should provide biochemically relevant signals and physical guidance cues, thus playing an active role in regeneration. To that end, laminin and laminin-polycaprolactone (PCL) blend nanofibers were fabricated to mimic peripheral nerve basement membrane. In vitro assays established 10% (wt) laminin content is sufficient to retain neurite-promoting effects of laminin. In addition, modified collector plate design to introduce an insulating gap enabled the fabrication of aligned nanofibers. The effects of laminin content and fiber orientation were evaluated in rat tibial nerve defect model. The lumens of conduits were filled with nanofiber meshes of varying laminin content and alignment to assess changes in motor and sensory recovery. Retrograde nerve conduction speed at 6 weeks was significantly faster in animals receiving aligned nanofiber conduits than in those receiving random nanofiber conduits. Animals receiving nanofiber-filled conduits showed some conduction in both anterograde and retrograde directions, whereas in animals receiving hollow conduits, no impulse conduction was detected. Aligned PCL nanofibers significantly improved motor function; aligned laminin blend nanofibers yielded the best sensory function recovery. In both cases, nanofiber-filled conduits resulted in better functional recovery than hollow conduits. These studies provide a firm foundation for the use of natural-synthetic blend electrospun nanofibers to enhance existing hollow nerve guidance conduits.

IL-23 Is Required for Neutrophil Homeostasis in Normal and Neutrophilic Mice

IL-23 is secreted by macrophages and dendritic cells in response to microbial products and inflammatory cytokines. IL-23 is a heterodimer composed of the unique IL-23p19 subunit linked to the common p40 subunit that it shares with IL-12. IL-23 is implicated in autoimmune diseases, where it supports the expansion of IL-17A-producing CD4+ Th17 cells. IL-23 also regulates granulopoiesis in a neutrostat regulatory feedback loop through IL-17A-producing neutrophil regulatory (Tn) cells, most of which express γδ TCR. This homeostatic system is disrupted in mice lacking adhesion molecules like β2-integrins (Itgb2−/−) which have defective neutrophil trafficking and neutrophilia. To test the role of IL-23 in the homeostatic regulation of circulating neutrophil numbers, we measured blood neutrophil numbers in p40-deficient (IL12b−/−) mice and found them reduced compared with wild-type mice. IL12b−/−Itgb2−/− mice, lacking β2-integrins, IL-12, and IL-23 showed significantly blunted neutrophilia compared with Itgb2−/− mice. Treatment of both IL12b−/− and IL12b−/−Itgb2−/− mice with IL-23, but not IL-12, restored circulating neutrophil counts. Serum levels of IL-17A were readily detectable in Itgb2−/− mice, but not in IL12b−/−Itgb2−/− mice, suggesting that IL-17A production is reduced when IL-23 is absent. Similarly, tissue mRNA expression of IL-17A was reduced in IL12b−/−Itgb2−/−mice compared with Itgb2−/− controls. The total number of CD3+ IL-17A-producing Tn cells were significantly reduced in the spleen and lamina propria of IL12b−/−Itgb2−/− mice, with the largest reduction found in γδ+ T cells. Our results suggest a prominent role of IL-23 in the regulation of granulopoiesis and the prevalence of IL-17A-producing Tn cells.

Protective roles of nitric oxide and testosterone in endotoxemia: evidence from NOS-2-deficient mice

Lipopolysaccharide (LPS)-induced septic shock, which triggers nitric oxide (NO) overproduction, multiple organ dysfunction, and death, can be affected by gender and sex hormones. We hypothesized that NO is beneficial during endotoxemia and that this beneficial effect is influenced by sex hormones. C57BL/6 wild-type (WT) mice and congenic inducible NO synthase knockout (KO) mice were injected with LPS, and mortality was recorded for 4 days. After 5 mg/kg LPS, female KO mice had significantly higher mortality than WT. After 12.5 mg/kg LPS, both male and female KO mice had significantly higher mortality than WT. Ovariectomy did not alter mortality, but orchiectomy dramatically increased mortality in KO mice. After 5 mg/kg LPS, exogenous testosterone completely prevented the increased mortality in KO female and orchiectomized KO male mice. WT survival was not affected by exogenous testosterone. After 12.5 mg/kg LPS, exogenous testosterone significantly improved survival of female KO mice. Serum enzymes and organ edema, which may not correlate with mortality, were significantly and similarly increased in both WT and KO endotoxemic mice; however, edema was not observed in KO hearts. Thus, NO plays a protective role in endotoxemia while having differential effects on different organs. Importantly, testosterone is beneficial in endotoxemia when NO production is deficient, and may be therapeutic in certain septic patients.Lipopolysaccharide (LPS)-induced septic shock, which triggers nitric oxide (NO) overproduction, multiple organ dysfunction, and death, can be affected by gender and sex hormones. We hypothesized that NO is beneficial during endotoxemia and that this beneficial effect is influenced by sex hormones. C57BL/6 wild-type (WT) mice and congenic inducible NO synthase knockout (KO) mice were injected with LPS, and mortality was recorded for 4 days. After 5 mg/kg LPS, female KO mice had significantly higher mortality than WT. After 12.5 mg/kg LPS, both male and female KO mice had significantly higher mortality than WT. Ovariectomy did not alter mortality, but orchiectomy dramatically increased mortality in KO mice. After 5 mg/kg LPS, exogenous testosterone completely prevented the increased mortality in KO female and orchiectomized KO male mice. WT survival was not affected by exogenous testosterone. After 12.5 mg/kg LPS, exogenous testosterone significantly improved survival of female KO mice. Serum enzymes and organ edema, which may not correlate with mortality, were significantly and similarly increased in both WT and KO endotoxemic mice; however, edema was not observed in KO hearts. Thus, NO plays a protective role in endotoxemia while having differential effects on different organs. Importantly, testosterone is beneficial in endotoxemia when NO production is deficient, and may be therapeutic in certain septic patients.

Oxyhemoglobin-induced cytotoxicity and arachidonic acid release in cultured bovine endothelial cells.

Background and Purpose An impairment of endothelial function is associated with vasospasm after subarachnoid hemorrhage. Oxyhemoglobin is considered to be a critical trigger in the pathogenesis of vasospasm. The present studies examined the direct effects of oxyhemoglobin on cultured endothelial cells from bovine carotid artery. Methods Confluent endothelial cells were treated with oxyhemoglobin, and the following were studied: 1) cell morphology, 2) cell density, and 3) the release of radiolabel from [3H] arachidonic acid-treated cells. Results Endothelial cells exposed to oxyhemoglobin exhibited detachment vacuoles, and cell density was significantly decreased in time- and dose-dependent manners. Superoxide dismutase, a free radical scavenger, provided partial protection against the cytotoxic effects of oxyhemoglobin. The release of radiolabel from [3H] arachidonic acid-treated cells was increased by oxyhemoglobin in time- and dose-dependent manners. Treatment with an inhibitor of phospholipase A2 or a calcium chelator inhibited the effects of oxyhemoglobin on arachidonic acid release and cellular viability. Conclusions Oxyhemoglobin exerts a direct cytotoxic effect on cultured endothelial cells, and this effect is associated with increased release from [3H] arachidonic acid-labeled cells. Phospholipase A2 and free radicals appear to participate in the pathogenesis of endothelial cell damage. Oxyhemoglobin-induced compromise of endothelial cells may contribute to cerebrovascular pathology.

Endoscopic Ultrasound-Guided Radiofrequency Ablation (EUS-RFA) of the Pancreas in a Porcine Model

Backgrounds. Limited effective palliative treatments exist for pancreatic cancer which includes surgery or chemotherapy. Radiofrequency ablation (RFA) uses high frequency alternating current to ablate diseased tissue and has been used to treat various tumors. In this study, we evaluated a prototype probe adjusted to the EUS-needle to perform EUS-RFA to permit coagulative necrosis in the pancreas. Methods. Five Yucatan pigs underwent EUS-guided radiofrequency ablation of the head of their pancreas. Using an EUS-needle, RFA was applied with 6 mm and then 10 mm of the probe exposed at specific wattage for preset durations. Results. Only one pig showed moderate levels of pancreatitis (20% proximal pancreatitis). The other animals showed much lower areas of tissue damage. In 3 of the 5 pigs, the proximal pancreas showed greater levels of tissue injury than the distal pancreas, consistent with the proximity of the tissue to the procedure site. In 1 pig, both proximal and distal pancreas showed minimal pancreatitis (1%). There was minimal evidence of fat necrosis in intra-pancreatic and/or extra-pancreatic adipose tissue. Conclusion. EUS-guided RFA of the pancreatic head with the monopolar probe through a 19-gauge needle was well tolerated in 5 Yucatan pigs and with minimal amount of pancreatitis.

Bispecific Monoclonal Antibodies Mediate Binding of Dengue Virus to Erythrocytes in a Monkey Model of Passive Viremia

Dengue viruses (DEN), causative agents of dengue fever (DF) and more severe dengue hemorrhagic fever (DHF)/dengue shock syndrome, infect over 100 million people every year. Among those infected, up to one-half million people develop DHF, which requires an extensive hospital stay. Recent reports indicate that there is a significant correlation between virus titer in the bloodstream of infected individuals and the severity of the disease, especially the development of DHF. This suggests that if there is a procedure to reduce viremia in infected subjects, then the severity of the disease may be controlled during the critical early stages of the disease before it progresses to DHF. We have generated bispecific mAb complexes (heteropolymer(s), HP), which contain a mAb specific for the DEN envelope glycoprotein cross-linked with a second mAb specific for the primate E complement receptor 1. These HP facilitate rapid binding of DEN to human and monkey E in vitro, with ∼90% bound within 5 min. Furthermore, in a passive viremia monkey model established by continuous steady state infusion of DEN, injection of HP during the steady state promoted rapid binding of DEN to the E, followed by subsequent clearance from the vascular system. Moreover, HP previously infused into the circulation is capable of efficiently capturing a subsequent challenge dose of DEN and binding it to E. These data suggest that HP potentially can be useful for alleviating DEN infection-associated symptoms by reducing titers of free virus in the vascular system.

Targeting of Pseudomonas aeruginosa in the Bloodstream with Bispecific Monoclonal Antibodies

We examined the ability of a bispecific mAb reagent, consisting of a mAb specific for the primate erythrocyte complement receptor cross-linked with an anti-bacterial mAb, to target bacteria in the bloodstream in an acute infusion model in monkeys. In vitro studies demonstrated a variable level of complement-mediated binding (immune adherence) of Pseudomonas aeruginosa (strain PAO1) to primate E in serum. In vivo experiments in animals depleted of complement revealed that binding of bacteria to E was <1% before administration of the bispecific reagent, but within 5 min of its infusion, >99% of the bacteria bound to E. In complement-replete monkeys, a variable fraction of infused bacteria bound to E. This finding may have significant implications in the interpretation of animal models and in the understanding of bacteremias in humans. Treatment of these complement-replete monkeys with the bispecific reagent led to >99% binding of bacteria to E. Twenty-four-hour survival studies were conducted; several clinical parameters, including the degree of lung damage, cytokine levels, and liver enzymes in the circulation, indicate that the bispecific mAb reagent provides a degree of protection against the bacterial challenge.

B Cell Complement Receptor 2 Transfer Reaction

The B cell C receptor specific for C3dg (CR2) shares a number of features with the primate E C receptor (CR1). Previously, we have demonstrated, both in vitro and in animal models, that immune complexes (IC) bound to primate E CR1, either via C opsonization or by means of bispecific mAb complexes, can be transferred to acceptor macrophages in a process that also removes CR1 from the E. We have now extended this paradigm, the transfer reaction, to include B cell CR2. We used both flow cytometry and fluorescence microscopy to demonstrate that IC bound to Raji cell CR2, either via C opsonization or through the use of an anti-CR2 mAb, are transferred to acceptor THP-1 cells. This reaction, which appears to require Fc recognition of IgG bound to Raji cell CR2, also leads to transfer of CR2. Additional support for the B cell transfer reaction is provided in a prototype study in a monkey model in which IC bound to B cell CR2 are localized to the spleen. These findings may have important implications with respect to defining the role of C in IC handling during the normal immune response.